The overall objective of this proposal is to characterize the structure and function of human centromeres, which are the fundamental chromosome components that ensure proper chromosome segregation during cell division. Errors in chromosome segregation result in aneuploidy and birth defects, and neoplasias. Neocentromeres are a clinically ascertained human centromere variant that provide mitotic stability to chromosomal fragments that have separated from endogenous centromeres. They have formed on single or low copy genomic DNA and do not contain the tandemly repeated alpha satellite DNA found at all endogenous human centromeres. The formation of neocentromeres on complex low-copy genomic DNA permits the structural and functional dissection of human centromeres onto the underlying linear DNA sequences. The following three Specific Aims will investigate human centromeres by 1) identification of the DNA sequences found at a collection of 12 neocentromeres that have formed in chromosome 13q, 2) use of neocentromeres to investigate the chromatin domain structure and minimal DNA sequences required for human centromere function, 3) analysis of the DNA sequence and chromosomal context at multiple independent neocentromeres from the same chromosomal location to investigate the requirements for human centromere formation. Investigation of human centromere structure, function and formation will greatly facilitate the construction of artificial human chromosomes for use as autonomous gene expression vectors.